The present invention relates to a method for producing (S)-4-hydroxy-2-ketoglutaric acid and to methods for producing compounds which can be formed from a precursor (S)-4-hydroxy-2-ketoglutaric acid, e.g., compounds such as (2S,4S)-4-hydroxy-L-glutamic acid and (2S,4S)-4-hydroxy-L-proline. (2S,4S)-4-hydroxy-L-proline has biological activities including anti-tumor cell activity [Cancer Res.48., 2483(1988)] and anti-mast cell activity (Japanese Unexamined Patent Publication No. 63-218621). (S)-4-hydroxy-2-ketoglutaric acid and (2S,4S)-4-hydroxy-L-glutamic acid are useful for the production of (2S,4S)-4-hydroxy-L-proline.
As a conventional method for producing (S)-4-hydroxy-2-ketoglutaric acid, a number of methods have been known, including a chemical deamination of threo-4-hydroxy-L-glutamic acid [Methods in Enzymology, 17 part B, 275].
The present inventors previously disclosed a method for producing (S)-4-hydroxy-2-ketoglutaric acid (Japanese Unexamined Patent Publication No. 7-289284), comprising allowing (e.g., providing) a biocatalyst, having activity to generate (S)-4-hydroxy-2-ketoglutaric acid from pyruvic acid, to act on glyoxylic acid and pyruvic acid or a compound capable of being converted into pyruvic acid through the action of the biocatalyst. Compared with the methods conventionally known, the method is far more industrially advantageous., but the method is disadvantageous in that the accumulation of (S)-4-hydroxy-2-ketoglutaric acid is less if inexpensive glucose is used as the substrate, and that expensive pyruvic acid should necessarily be used as the substrate so as to yield an accumulation level of (S)-4-hydroxy-2-ketoglutaric acid above 20 mM.
The following conventional methods for producing (2S,4S)-4-hydroxy-L-glutamic acid have been known; a method comprising allowing glutamate dehydrogenase to act on chemically synthesized DL-4-hydroxy-2-ketoglutaric acid in the presence of ammonia and NADPH and separating the resulting 4(R)- and 4(S)-4-hydroxy-glutamic acid by ion exchange chromatography; a method comprising extracting (2S,4S)-4-hydroxy-L-glutamic acid from a plant (Phlox decussata) [Methods in Enzymology, 17 part B, 277]; and a method comprising allowing transaminase to act on L-4-hydroxy-2-ketoglutaric acid and cysteine sulfinic acid [Tetrahedron Letters, 28, 1277 (1987)].
The present inventors have previously disclosed a method for producing (2S,4S)-4-hydroxy-L-glutamic acid, comprising allowing (e.g., providing) a biocatalyst, having activity to generate (2S,4S)-4-hydroxy-L-glutamic acid from pyruvic acid and glyoxylic acid in the presence of an amino group donor, to act on glyoxylic acid and pyruvic acid or a compound capable of being converted into pyruvic acid (Japanese Unexamined Patent Publication No. 8-80198). The method is industrially advantageous in that only the 4(S) form can be produced; however, the method is laborious and disadvantageous in that the method further requires a step of converting (S)-4-hydroxy-L-ketoglutamic acid into (2S,4S)-4-hydroxy-L-glutamic acid by adding another bacterium to (S)-4-hydroxy-L-ketoglutamic acid after the step of synthesis of (S)-4-hydroxy-L-ketoglutamic acid so as to produce a great amount of (2S,4S)-4-hydroxy-L-glutamic acid by the method.
As a conventional method for producing (2S,4S)-4-hydroxy-L-proline, the following methods have been known; a method comprising culturing a microorganism of genus Helicoceras or Acrocylindrium and extracting proline from the culture (Japanese Unexamined Patent Publication No. 5-111388); and a method comprising allowing (e.g., providing) a microorganism, having activity to convert 4-hydroxy-2-ketoglutaric acid into 4-hydroxy-L-proline, to act on 4-hydroxy-2-ketoglutaric acid (Japanese Unexamined Patent Publication No. 3-266996); and the like. However, the industrial application of these methods is difficult, because the yield of the former method is low and the latter method requires laborious procedures for separation and purification of the simultaneously generated 4(S) form and 4(R) form.
An object of the present invention is to provide a method for industrially advantageously producing (S)-4-hydroxy-2-ketoglutaric acid and compounds produced from the precursor (S)-4-hydroxy-2-ketoglutamic acid, for example (2S,4S)-4-hydroxy-L-glutamic acid and (2S,4S)-4-hydroxy-L-proline.
The present invention relates to a method for producing an optically active compound, comprising allowing (e.g., providing) a recombinant microorganism, carrying recombinant DNA including a DNA fragment encoding (S)-4-hydroxy-2-ketoglutarate aldolase (abbreviated as xe2x80x9cKAL genexe2x80x9d hereinbelow), to act on sugar and glyoxylic acid in the presence or absence of an amino group donor in an aqueous medium and collecting optically active (S)-4-hydroxy-2-ketoglutaric acid generated in the aqueous medium or a compound produced from the precursor (S)-4-hydroxy-2-ketoglutaric acid (abbreviated as xe2x80x9c4(S)KHGxe2x80x9d hereinbelow).